PROJECT SUMMARY The sense of hearing depends on the transmission of auditory information from the spiral ganglion neurons (SGNs) of the auditory nerve to the brain. Following cochlear injury, SGNs can become damaged or lose axonal connectivity to their targets, resulting in sensorineural hearing loss (SNHL). Processes of axon elimination are seen during development of the auditory nerve when SGNs undergo a critical period of maturation where excessive neural cells and nerve fibers are eliminated and refined to obtain innervation patterns seen in the adult cochlea. This proposal aims to elucidate the cellular and molecular mechanisms governing developmental auditory nerve refinement to understand mechanisms that contribute to adult auditory nerve refinement. Preliminary data shows that cochlear macrophages are actively phagocytosing glial cells and auditory nerve fibers in the postnatal auditory nerve, suggesting that macrophages are required for auditory nerve refinement. Complement signaling is essential for regulating macrophage-related immune functions and contributes to the maturation and refinement of neural tissues in the central nervous system. Preliminary studies of auditory nerves at postnatal day 7 revealed increased expression of alternative complement genes, suggesting that alternative complement signaling contributes to auditory nerve development. Our overarching hypothesis is that alternative complement signaling promotes auditory nerve refinement by stimulating cochlear macrophages to eliminate excessive auditory glia and nerve fibers. Specific Aim 1 tests the hypothesis that the elimination of supernumerary fibers and auditory glia by macrophages is necessary for hearing onset in the postnatal cochlea. Activated macrophages will be depleted genetically to test how they affect hearing onset and auditory nerve development. Specific Aim 2 tests the hypothesis that alternative complement pathway signaling mediates macrophage activity during auditory nerve refinement in the postnatal mouse cochlea. Auditory nerve refinement and hearing onset will be evaluated in mice deficient in alternative complement pathway signaling. Findings from this study will have impact on understandings of auditory nerve development and definitively test the role of cochlear macrophages in achieving mature neural architecture and hearing function. This study will identify molecules that play regulatory roles in auditory nerve refinement, and thus present therapeutic targets for developmental abnormalities and/or adult stage hearing dysfunction. The training and mentorship plan described in this proposal will prepare the candidate for a successful career as an independent scientist. Relevance to Agency: Results from this study will shed light on how the auditory nerve develops and is maintained. This work proposes that regulation of cochlear macrophages and alternative complement signaling is a potential target for treatment of hearing loss and other nerve pathologies.